Cell analysis plays an important role in understanding life processes and biological mechanisms. Microfluidic chip structures are designed to be flexible, low-cost, and easy to integrate and automate, expanding the way cell biology is studied as a cell analysis tool. The manufacturing process of traditional microfluidic chips is complex and expensive, which limits the application of microfluidic chips in cell biology.
Using flexible, direct and rapid prototyping 3D printing technology to fabricate microfluidic chips, a series of small cell analysis platforms have been constructed by combining 3D printed microfluidic chips with biocompatible paper chips and PDMS membranes, which can be used for cell-based analysis of compound activity. To study the effect of oxygen and dissolved oxygen concentration gradient on cells and its mechanism of action.
Cell-based compound activity
Configuring and dispensing different concentrations of stimuli is cumbersomeCells are grown in 2D in multi-well plates and cannot mimic 3D growth in vivo;Multi-well plates are an open culture system, and the concentration of volatile substances can vary over time, creating uncertainty in the results.
We have jointly built a cell analysis platform with a 3D printed microfluidic chip and a paper chip, the 3D printing chip consists of two layers, the upper layer is the concentration gradient cambium layer embedded in the "Christmas tree" network structure, the lower layer is the cell culture layer with the culture chamber, and the paper chip is placed in the lower culture chamber of the 3D printed chip as the cell culture carrier.
The upper effluent provides continuous and stable concentration gradient stimulation to the cells in the lower layer. The structural design of the upper and lower layers separates the concentration gradient cambium layer from the cell culture layer, which not only facilitates the cultivation of cells in the chip, but also prevents the pipes of cells trapped outside the culture chamber from blocking the flow channel. Paper chips are used as a medium for cell culture and analysis.
It can realize a three-dimensional cell growth method similar to that in vivo, saving the amount of reagents used for cell staining analysis. The flow rate of the liquid injection is 2At 5 l min, the 3D printed microfluidic chip could form a good concentration gradientThe results of cell culture showed that the cells cultured on this platform had good growth viability and cell morphology.
Hydrogen sulfide is a gas signaling molecule inside the cell
The effect of H2S on the proliferative activity of tumor cells has been controversial, and related H2S experiments are usually performed in multi-well plates, and in an open multi-well plate cell culture system, H2S is easily volatile, affecting the stability and reliability of experimental results.
We investigated the effect of continuous low concentrations of H2S on sMMC-7721 in hepatocellular carcinoma cells on a constructed 3D printed microfluidic concentration gradient chip and paper chip composite platform. The results showed that low concentrations of H2S continuously acted on tumor cells to inhibit their proliferation by inducing apoptosis, and the mechanism of action was as follows: after H2S entered tumor cells.
It is used with intracellular NO to form biologically active polysulfide intermediates, induce a series of physiological changes in tumor cells, and eventually lead to apoptosis. In order to study the physiological response of cells in a hypoxic environment, hypoxic perfusion chambers are used to study the environmental hypoxia caused by pollution.
Hypoxic workstations are used to create a hypoxic environment, but these devices** are expensive and do not provide a physiological hypoxic gradient. We injected air and nitrogen into the 3D printing microfluidic chip at the same time to generate an oxygen concentration gradient, and combined with the cellulose film paper chip as the substrate for cell culture in the 3D printing chip, we constructed an oxygen gradient cell analysis platform.
The platform produces a stable linear oxygen concentration gradient over a short period of time. The highly biocompatible NC film paper chip serves as the substrate for cell culture and analysis in 3D printed chips, and unlike traditional PDMS oxygen concentration gradient cell culture chips that rely only on fluorescence imaging, cells processed on this platform can be processed by fluorescence imaging and flow cytometry.
Western blot, qPCR, and other methods were used for analysis. The effects of oxygen gradient on zebrafish cell cycle, intracellular signaling molecules and gene expression were studied on this platform using zebrafish embryonic cell lines as a model. After zebrafish cells were cultured on an oxygen gradient platform, the oxygen level within the cells also showed a gradient change.
Hypoxia causes an increase in reactive oxygen species within zebrafish cells and allows the hypoxia-inducible factor HIF-1 to accumulate in the cells;HIF-1 further stimulates intracellular vascular endothelial growth factor gene transcription to generate more blood vessels and increase oxygen supplyAt the same time, hypoxia arrests zebrafish cell cycle progression at G0 G1.
Cell cycle arrest causes cells to stop proliferating to maintain cell viability under hypoxic conditions. Hypoxia in water bodies has become a serious environmental problem, and in order to study the effects of hypoxia on aquatic animals, there is an urgent need for a simple hypoxic platform. We use 3D printing technology to make microfluidic chips in one step.
Two liquids with different dissolved oxygen concentrations can generate a dissolved oxygen gradient containing multiple dissolved oxygen concentrations through the "Christmas tree" structure pipe network of 3D printed chips, and the transparent and biocompatible PDMS membrane can be used as a substrate for cell culture, and the embryos and larvae of aquatic animals can be directly cultured in the growth chamber of the 3D printed chip.
Receive cultures with different concentrations of dissolved oxygen. It has been verified that the dissolved oxygen concentration gradient generated in the 3D printing chip has a good linear correlation with the theoretical dissolved oxygen concentrationThe platform has good biocompatibility, and zebrafish cells and embryos are in good condition for a long time inside it. On the constructed DO gradient platform, the embryos are incubated for a short time.
Zebrafish with transparent bodies were used as model organisms to study the effects of different dissolved oxygen concentrations on zebrafish at the cellular, embryonic and larval levels. It was found that hypoxia caused reactive oxygen species to be produced in zebrafish cells, embryos, and larvae, which ultimately led to apoptosis and developmental impairmentHypoxic stress forces an increase in intracellular NO content in zebrafish.
NO may be a protective molecule produced by fish cells in a hypoxic environment to counteract the adverse effects of hypoxia;Hypoxia reduces the hatchability of zebrafish embryos while suppressing the embryos' heart rate with no significant effect on the juveniles' heart rate.
Cells are the basic unit of life activities
The study of life activities at the cellular level can lead to more nuanced and truthful information.
With the deepening of life science research, in recent years, the study of cells has penetrated to the molecular level, not only in terms of morphology, the submicroscopic, ultramicroscopic and molecular structure of each part of the cell, but also the physiological function of its chemical composition, information transmission and other life activities, in order to explore the basic laws of life process.
The microfluidic chip has a channel size in the micron to millimeter range, matching the volume of animal cells, and is suitable for manipulation and detection of individual cells within the channelThe internal channels of the microfluidic chip can form a relatively closed environment similar to that of an organism;The small channel size not only reduces sample consumption, but also speeds up mass and heat transfer.
Helps to improve the sensitivity of cell analysis;The flexible design of the microfluidic chip allows multiple cell research units and multiple cell detection methods to be integrated to achieve high-throughput and comprehensive cell research. So far, microfluidic chips have been widely used in many fields such as cell culture, cell sorting, drug screening, tumor research, tissue and organ engineering, etc.
In the field of biochemical analysis, especially in the field of cell analysis, the use of various instruments and devices to simulate the physiological environment of organisms such as temperature, biological factor concentration and mechanical force, and the detection and analysis of cells in them will help people deeply understand and study various biological phenomena, therefore, the establishment of a cell analysis platform that simulates the physiological environment in vitro will promote cell biology.
Development of medicine and other related disciplines. The microfluidic chip-based cell analysis platform can effectively simulate the microenvironment of cells in organisms, which is of great significance for the in-depth study of biological phenomena and the development of cell biology.
Microfluidic chip, also known as micro-total analysis system or laboratory-on-a-chip, is a miniature device that integrates or partially integrates basic operating units such as sample pretreatment, reaction, separation, cell culture, sorting, lysis, and detection. Microchannels form a network structure that runs through the entire system with controlled fluids, and have some of the functions of a conventional biological or chemical laboratory.
The concept of microfluidic chip was proposed by Manz in the early 90s of the 20th century, and after more than 30 years of development, microfluidic chip technology has been widely used in biochemical analysis, high-throughput drug screening, point-of-care diagnosis and other biochemical and medical research.
In 2011, the United States launched the "Microbio Shandong Normal University Doctoral Degree Management System" based on microfluidic chips to ensure the leading position in the field of new drug discovery in the United States. It is believed that the project can greatly reduce the cost and cycle of new drug discovery, which also marks the recognition of microfluidic chips by academia and industry at a higher level and on a larger scale.
Microfluidic chip materials
The material of microfluidic chip is the carrier of the development of microfluidic chip, and it is of great significance to realize the various functions of microfluidic chip. Early microfluidic chips were mainly made of glass.
With the invention of new materials and the development of manufacturing technology, more materials are used in the production of microfluidic chips. At present, the main materials of microfluidic chips can be divided into three categories: inorganic materials, polymer materials and paper-based materials. Silicon was the first inorganic material to be used in the fabrication of microfluidic chips.
Silicon has good thermal conductivity and ensures uniform temperature distribution in the deviceThe surface of silicon is easily chemically modified, and the exposed silica hydroxyl groups on its surface can be combined with different types of silanization reagents, thereby changing the hydrophilicity and hydrophobicity of different regionsSilicone materials are chemically inert and resistant to organic solvents, allowing for high-precision microchannels to be machined on the surface.
Silicon materials also have obvious drawbacks, such as: poor elasticity, fragility, and difficulty in integrating fluid control units such as micropumps and microvalves on its surface;The material has poor transparency and cannot be combined with optical testing methods such as fluorescence detection and image acquisition, which limits its application in the field of bioanalysis. Glass, as another widely used inorganic chip material, has unique advantages.
The glass has good light transmittance, low self-fluorescence background, and can be observed in real timeThe surface of the glass has less non-specific adsorption, and the hydroxyl group on its surface is biomodified, which can be used for the detection of biomoleculesThe glass has a high electroosmotic mobility and is driven by an electroosmotic fluid, which allows the sample to be injected without an external pump valve and enables rapid separation of the mixture.
Inorganic materials are not the mainstream materials of microfluidic chips, the main reason is that each inorganic material chip has to be made from scratch, and the reuse of the chip is low, and at the same time, the surface processing of inorganic materials microchannels requires dangerous chemicals such as hydrofluoric acid;The sealing of inorganic chips requires a high-temperature, high-pressure environment.
Polymers have been used in the production of microfluidic chips since 2000. There are many types of polymer materials, which can be modified and modified, so they have a high degree of flexibility in selectionThe processability and plasticity of polymers are good, the synthetic cost is low, and it is suitable for mass production. Silicone elastomer materials are currently the preferred materials for making microfluidic chips.
Represented by polydimethylsiloxane, liquid PDMS monomer, after adding a polymerization initiator, polymerizes at 40-70 to form a solid material. PDMS has good elastic performance and can integrate control units such as micro pumps and micro valvesCompared to other chip materials, PDMS has gas permeability.
It is beneficial for its application in the field of cell analysis;In addition, PDMS has good light transmittance, which allows the chip to be combined with various microscopy techniques to observe cells in real time. Based on the above advantages, PDMS can be widely used in biochemical analysis fields such as cell culture and cell sorting. PDMS also has certain limitations due to its intolerance to organic solvents.
The application range is limited to aqueous solution systems;The hydrophobicity of the material surface can easily cause non-specific adsorption of drug molecules and biomoleculesThe high gas permeability of PDMS will cause the volatilization of water molecules and change the concentration of the solution in the chip, making it unsuitable for the creation of gas-related microenvironment chips.
Conclusion
Thermosetting molecules will be cross-linked and cured by heating or radiation to form a rigid network-like structure, and the curing process is irreversible, representing substances such as Su-8 photoresist, polyimide, epoxy plastic, etc., which have high temperature resistance.
Organic solvent resistance and good light transmittance, but this kind of material has obvious rigid characteristics after curing, and it is difficult to integrate functional units such as micropumps and microvalves similar to inorganic materials. Thermoplastic materials can be reshaped at high temperatures and can be shaped repeatedly, and are solid at room temperature, when their glass phase transition temperature is reached, the thermoplastic material softens and can be molded.
Representative thermoplastic materials are polymethyl methacrylate, polycarbonate, polystyrene, etc., the thermoplastic material is poor, the rigidity is large after shaping, and the micro-valve, micro-pump, etc. can not be integrated on the chip, but the thermoplastic material can be mass-produced in a short period of time through thermoshaping, the processing cost is low, and it can be used in a large number of commercial products.